The Animal By-Products (Wales) Regulations 2006 (revoked)

Regulation 158 19(1)

SCHEDULE 1 E+W Additional requirements for biogas and composting plants

PART I E+W Premises

1.—(1) There must be– E+W

(a) a reception area in which untreated animal by-products (including catering waste) are received;

(b) an area in which vehicles and containers are cleansed and disinfected with adequate facilities for doing this; and

(c) a clean area in which treated compost or digestion residues are stored.

(2) The clean area must be adequately separated from the reception area and the area in which vehicles and containers are cleansed and disinfected so as to prevent contamination of the treated material. Floors must be laid so that liquid cannot seep into the clean area from the other areas.

(3) The reception area must be easy to clean and disinfect and must have an enclosed and lockable place or container to receive and store the untreated animal by-products.

2.  The animal by-products must be unloaded in the reception area and either– E+W

(a) treated immediately; or

(b) stored in the reception area and treated without undue delay.

3.  The plant must be operated in such a way that– E+W

(a) treated material is not contaminated by untreated or partially treated material or liquids arising from it; and

(b) partially treated material is not contaminated with material which has not been treated to the same extent or liquids arising from it.

4.  The operator must identify, control and monitor suitable critical points in the operation of the plant to demonstrate that– E+W

(a) these Regulations and the Community Regulation are complied with;

(b) treated material is not contaminated by untreated or partially treated material or liquids arising from it; and

(c) partially treated material is not contaminated with material which has not been treated to the same extent or liquids arising from it.

5.  Containers, receptacles and vehicles used for transporting untreated animal by-products must be cleaned in the dedicated area before they leave the premises and before any treated material is loaded. In the case of vehicles transporting only untreated catering waste and not subsequently transporting treated material, only the wheels of the vehicle need be cleaned. E+W

PART II E+W Treatment systems and parameters for catering waste

1.  Unless an approval specifically permits a different system, catering waste must be treated by one of the systems specified in the table below. The system must ensure that the material is treated to the following parameters: E+W

Composting

System	Composting in a closed reactor	Composting in a closed reactor	Composting in housed windrows
Maximum particle size	40cm	6cm	40cm
Minimum temperature	60°C	70°C	60°C
Minimum time spent at the minimum temperature	2 days	1 hour	8 days (during which the windrow must be turned at least 3 times at no less than 2 days intervals)

The time temperature requirements must be achieved as part of the composting process.

Biogas

System	Biogas in a closed reactor	Biogas in a closed reactor
Maximum particle size	5cm	6cm
Minimum temperature	57°C	70°C
Minimum time spent at the minimum temperature	5 hours	1 hour

2.  The approval must normally specify one of the methods in the table, but the National Assembly may approve a different system if it is satisfied that it achieves the same reduction in pathogens as those methods (including any additional conditions imposed on those methods) in which case the approval must fully describe the whole system. E+W

Composting plants E+W

3.  If the approval for a composting plant specifies one of the methods in the table, it must specify which one and, in addition, must have as a condition either that–

(a) measures must be taken at source to ensure that meat was not included in the catering waste and that following treatment the material is stored for at least 18 days (storage need not be in an enclosed system), or

(b) following the first treatment, the material must be treated again using one of the methods in the table and specified in the approval (not necessarily the same method as was used for the first treatment) except that, if the treatment is in a windrow, the second treatment need not be in a housed windrow.

Biogas plants E+W

4.  The approval for a biogas plant must specify one of the methods in the table and in addition require that either–

(a) measures were taken at source to ensure that meat was not included in the catering waste; or

(b) following treatment the material is stored for an average of 18 days.

Regulation 17(4)

SCHEDULE 2 E+W Ruminant derived fluid

Treatment or discharge of fluid from processing ruminant by-products E+W

1.—(1) Any person who processes any ruminant animal by-product must–

(a) discharge or consign for discharge the fluid arising from the processing into–

(i) a public sewer in accordance with a trade effluent consent or agreement from the relevant sewerage undertaker under the Water Industry Act 1991 M1 ; or

(ii) controlled waters (within the meaning of the Water Resources Act 1991 M2 ) in compliance with a discharge consent from the Environment Agency under that Act; or

(b) treat the fluid arising from the processing on the processing premises in such a way that the treated fluid has–

(i) a level of suspended solids of no more than 80 mg/litre, and

(ii) a biochemical oxygen demand of no more than 60 mg/litre,

and failure to do so is an offence.

(2) If the person who processes the animal by-products consigns fluid that has not been treated in accordance with sub-paragraph (1)(b) for discharge by another person, and that person fails to discharge it in accordance with sub-paragraph (1)(a)–

(a) both that person and the processor are guilty of an offence; but

(b) it is a defence for the processor to show that the processor believed on reasonable grounds that that person would discharge the fluid in accordance with sub-paragraph 1(a).

(3) This paragraph does not apply in relation to blood that has not been mixed with any other ruminant material.

Marginal Citations

M1 1991 c. 56.

M2 1991 c. 57.

Measurement of treated fluid E+W

2.—(1) In order to ensure that the treated fluid complies with the levels in paragraph 1(1)(b) an operator who treats fluid in accordance with that paragraph must as a minimum carry out the following measurements.

(2) The operator must continuously monitor the level of suspended solids in the treated fluid or alternatively measure it three times a day.

(3) Once a week the operator must measure the level of suspended solids in the treated fluid by a method that conforms with “Suspended Settleable and Total Dissolved Solids in Waters and Effluents M3 ”.

(4) Once a week the operator must measure the biochemical oxygen demand of the treated fluid by a method which conforms with the “5 day Biochemical Oxygen Demand (BOD5) M4 ”.

(5) If any of these measurements demonstrates that the treated fluid does not comply with the levels in paragraph 1(1)(b) the operator must ensure that the operator only discharges the treated fluid in accordance with paragraph 1(1)(a) until further tests show that the treatment system is achieving the required levels.

(6) It is an offence to fail to comply with any provision of this paragraph.

Marginal Citations

M3 This is set out in the series “Methods for the Examination of Waters and Associated Materials” available on the Environment Agency internet web-page (http://www.environment-agency.gov.uk/nls) but previously published by HMSO as ISBN 011751957X.

M4 This is set out in the series “Methods for the Examination of Waters and Associated Materials” available on the Environment Agency internet web-page (http://www.environment-agency.gov.uk/nls) but previously published by HMSO as ISBN 0117522120.

Records E+W

3.—(1) Any person who processes any ruminant animal by-products must, as soon as is reasonably practicable, record the dates and results of the measurements taken in accordance with paragraph 2.

(2) For all fluid discharged or consigned from the processing premises that person must, as soon as is reasonably practicable, record–

(a) whether the fluid was treated or untreated;

(b) the date and method of the discharge or consignment;

(c) the quantity discharged or consigned;

(d) where it was discharged, or the premises to which it was consigned; and

(e) the name of the haulier, if any.

(3) It is an offence to fail to comply with any provision of this paragraph.

Records of consignment E+W

4.—(1) Any person who consigns any fluid arising from the processing of ruminant animal by-products (whether or not that person processed the by-products personally) from any premises must, as soon as is reasonably practicable, record–

(a) the address of the premises from which the fluid is collected;

(b) the date on which the fluid is collected;

(c) the quantity and description of the fluid, and whether treated or untreated;

(d) the place at which it is to be discharged or disposed of.

(2) He or she must give a copy to the person who transports the fluid.

(3) The transporter must keep his or her copy of the record with the consignment until the fluid is discharged or disposed of.

(4) The consignor must keep a copy of the record for at least two years, and the transporter must keep it for at least two years.

(5) It is an offence to fail to comply with any provision of this paragraph.

Regulation 21

SCHEDULE 3 E+W Testing Methods

PART I E+W METHOD FOR THE ISOLATION OF CLOSTRIDIUM PERFRINGENS

Time of testing E+W

1.  Tests must be begun on receipt of the sample or on the first working day which allows this method to be completed. If the test is not begun on the day of receipt the sample must be stored in a refrigerator at between 2°C and 8°C until required. If the sample has been refrigerated it must be removed from the refrigerator and stored at room temperature for at least one hour before the test is started.

Samples E+W

2.  Tests must be carried out using two 10 gram portions of each sample submitted for testing. Each 10 gram sample must be placed aseptically in a sterile container containing 90 ml Clostridium perfringens diluent consisting of 0.1% peptone and 0.8% sodium chloride at a pH of 7 and mixed thoroughly until the sample is evenly suspended.

Inoculations E+W

3.  For each portion of the sample 1 ml of solution must be transferred to a sterile 90 mm petri dish (in duplicate), to which 15 ml of Shahidi – Ferguson agar (SF agar) M5 at a temperature of 47°C±1°C must be added and immediately gently mixed by swirling the dish with 5 clockwise and 5 anticlockwise circular movements.

Marginal Citations

M5 Shahidi-Ferguson agar – See Shahidi, S. A. and Ferguson, A. R. (1971) Applied Microbiology 21:500-506.American Society for Microbiology, 1913 1 St N.W., Washington DC 20006, USA.

4.  Once the agar has set, each agar plate must be overlaid with a further 10 ml SF agar at a temperature of 47°C±1°C. Once the overlay has set and with the plate lids uppermost the plates must be incubated anaerobically at 37°C±1°C for 20 hours±2 hours. E+W

Samples with colonies of Clostridium perfringens E+W

5.  After incubation each set of duplicate plates must be examined for colonies characteristic of Clostridium perfringens (black). The sample provisionally fails if any colonies characteristic of Clostridium perfringens are present, in which case the following procedure must be followed to establish whether or not the colonies are Clostridium perfringens.

6.  In the case of each plate, 10 characteristic colonies of Clostridium perfringens must be subcultured on to a further SF agar plate. If there are less than 10 colonies on the plate, all characteristic colonies must be subcultured on to the further plate. The plates must be incubated anaerobically at 37°C±1°C for 20 hours±2 hours. E+W

7.  If the surface area of the plates is overgrown and it is not possible to select well isolated characteristic colonies, 10 suspect colonies must be subcultured on to duplicate SF agar plates and incubated anaerobically at 37°C±1°C for 20 hours±2 hours. E+W

8.  One characteristic colony from each plate must be subcultured on to SF agar and incubated anaerobically at 37°C±1°C for 20 hours±2 hours. E+W

Subcultured colonies E+W

9.  After incubation each plate must be examined for colonies characteristic of Clostridium perfringens. All colonies characteristic of Clostridium perfringens must be–

(a) stab inoculated into motility nitrate medium M6 ; and

(b) inoculated into either lactose gelatin medium M7 or charcoal gelatin discs M8 ;

and incubated anaerobically at 37°C±1°C for 20 hours±2 hours.

Marginal Citations

M6 Motility nitrate medium – See Hauschild AHW, Gilbert RJ, Harmon SM, O"Keefe MF, Vahlefeld R, (1997) ICMSF Methods Study VIII, Canadian Journal of Microbiology 23, 884-892. National Research Council of Canada, Ottawa ON K1A oR6, Canada

M7 Lactose gelatin medium – See Hauschild AHW, Gilbert RJ, Harmon SM, O"Keefe MF, Vahlefield R, (1997) ICMSF Methods Study VIII, Canadian Journal of Microbiology 23, 884-892.

M8 Charcoal gelatin discs – See Mackie and McCartney, (1996) Practical Medical Microbiology 14, 509. Churchill Livingstone, Robert Stevenson House, 1-3 Baxter"s Place, Leith Walk, Edinburgh EH1 3AF.

Motility E+W

10.  The motility nitrate medium must be examined for the type of growth along the stab line. If there is evidence of diffuse growth out into the medium away from the stab line, the bacteria must be considered to be motile.

Reduction of nitrate to nitrite E+W

11.  After examination of the motility nitrate medium, 0.2 ml to 0.5 ml of nitrite detection reagent must be added to it. The formation of a red colour confirms that the bacteria have reduced nitrate to nitrite. Cultures that show a faint reaction (i.e. a pink colour) should be discounted. If no red colour is formed within 15 minutes, a small amount of zinc dust must be added and the plate allowed to stand for 15 minutes. If a red colour is formed after the addition of zinc dust no reduction of nitrate to nitrite has taken place.

Production of gas and acid from lactose and liquefaction of gelatin E+W

12.  The lactose gelatin medium must be examined for the presence of small gas bubbles in the medium.

13.  The lactose gelatin medium must be examined for colour. A yellow colour indicates fermentation of lactose. E+W

14.  The lactose gelatin medium must be chilled for one hour at 2 – 8°C and then checked to see if the gelatin has liquefied. If the medium has solidified it must be re-incubated anaerobically for a further 18 – 24 hours, the medium chilled for a further one hour at 2 – 8°C and again checked to see if the gelatin has liquefied. E+W

15.  The presence of Clostridium perfringens must be determined on the basis of the results from paragraphs 10 to 14. Bacteria which produce black colonies on SF agar, are non-motile, reduce nitrate to nitrite, produce gas and acid from lactose and liquefy gelatin within 48 hours must be considered to be Clostridium perfringens. E+W

Control Tests E+W

16.  Control tests must be carried out each day that a test is initiated using–

(a) Clostridium perfringens no more than seven days old at the time of use;

(b) Escherichia coli NCTC 10418 M9 or equivalent not more than seven days old at the time of use; and

(c) processed animal protein or compost or digestion residue which is free of Clostridium perfringens.

Marginal Citations

M9 The National Collection of Type Cultures, Central Public Health Laboratory, 61 Colindale Ave, London NW9 5HT.

17.  10 gram portions of the rendered animal protein must be placed aseptically in each of two sterile containers containing 90 ml Buffered Peptone Water (BPW) M10 and mixed thoroughly until the samples are evenly suspended. E+W

Marginal Citations

M10 Buffered Peptone Water – See Edel, W. and Kampelmacher, E.H. (1973) Bulletin of World Health Organisation, 48: 167-174, World Health Organisation Distribution and Sales, CH-1211, Geneva 27, Switzerland (ISSN 0042-9686).

18.  One colony of Clostridium perfringens must be placed in 10 ml BPW and mixed to form an even suspension. 0.1 ml of the suspension must be added to the suspension in the preceding paragraph. This must be repeated for Escherichia coli. E+W

19.  These are then treated and examined in the same way as test samples. If no typical colonies are formed then that day's testing must be invalid and must be repeated. E+W

PART II E+W METHODS FOR THE ISOLATION OF SALMONELLA

A. E+W BACTERIOLOGICAL METHOD

1.—(1) Tests must be begun on receipt of the sample or on the first working day which allows this method to be completed. If the test is not begun on the day of receipt the sample must be stored in a refrigerator until required. If the sample has been refrigerated it must be removed from the refrigerator and stored at room temperature for at least four hours before the test is started. E+W

(2) Tests must be carried out in duplicate using two 25 gram portions of each sample submitted for testing.

Day one E+W

2.  On day one, each 25 gram sample must be placed aseptically in a container containing 225 ml Buffered Peptone Water (BPW) and incubated at 37°C±1°C for 18 hours±2 hours.

Day two E+W

3.  On day two, 0.1 ml from the container of incubated BPW must be inoculated into 10 ml Rappaports Vassiliadis broth (RV broth) M11 and incubated at 41.5°C±0.5°C for 24 hours ± 3 hours.

Marginal Citations

M11 Rappaports Vassiliadis Broth – See Vassiliadis P, Pateraki E, Papaiconomou N, Papadkis J A, and Trichopoulos D (1976) Annales de Microbiologie (Institut Pasteur) 127B: 195-200. Elsevier, 23 rue Linois, 75724 Paris, Cedex 15, France.

Day three E+W

4.  On day three, the RV broth must be plated out on to two 90 millimetre plates of Brilliant Green Agar (BGA) M12 or on to one 90 millimetre plate of BGA and one 90 millimetre plate of Xylose Lysine Deoxycholate Agar (XLD) M13 using a 2.5 mm diameter loop. The plates must be inoculated with a droplet taken from the edge of the surface of the fluid by drawing the loop over the whole of one plate in a zig zag pattern and continuing to the second plate without recharging the loop. The space between the loop streaks must be 0.5 cm – 1.0 cm. The plates must be incubated at 37°C ±2°C for 24 hours ± 3 hours.

Marginal Citations

M12 Brilliant Green Agar – See Edel W and Kampelmacher E H (1969) Bulletin of World Health Organisation 41:297-306, World Health Organisation Distribution and Sales, CH-1211, Geneva 27, Switzerland (ISSN 0042-9686).

M13 Xylose Lisene Deoxycholate Agar – See Taylor W I, (1965) American Journal of Clinical Pathology, 44:471-475, Lippincott and Raven, 227E Washington Street, Philadelphia PA 19106, USA.

5.  The residual RV broth must be reincubated at 41.5°C±0.5°C for a further 24 hours. E+W

Day four E+W

6.  On day four the plates must be examined and a minimum of 3 colonies from each plate showing suspicion of Salmonella growth must be subcultured–

(a) on to a blood agar plate;

(b) on to a MacConkey agar plate M14 ; and

(c) into biochemical media suitable for the identification of Salmonella.

Marginal Citations

M14 MacConkey agar – See (1963) International Standards for Drinking Water, World Health Distribution and Sales, CH-1211, Geneva 27, Switzerland.

These media must be incubated at 37°C overnight. E+W

7.  The reincubated RV broth must be plated out as described in paragraph 4.

Day five E+W

8.  On day five the incubated composite media or equivalent must be examined and the findings recorded, discarding cultures which are obviously not Salmonella. Slide serological tests must be performed using Salmonella polyvalent “O”and polyvalent “H” (phase 1 and 2) agglutinating sera on selected suspect colonies collected from the blood agar or MacConkey plates. If reactions occur with one or both sera, the colonies must be typed by slide serology. If requested in writing by the National Assembly , the operator of the laboratory must send a subculture to a Regional Veterinary Laboratory of the Veterinary Laboratories Agency of the Department for Environment, Food and Rural Affairs for further typing.

9.  The plates referred to in paragraph 7 must be examined and further action taken as in paragraph 6 and 8. E+W

B. E+W ELECTRICAL CONDUCTANCE METHOD

10.  Tests must be begun on receipt of the sample or on the first working day which allows the following method to be completed. If the test is not begun on the day of receipt the sample must be stored in a refrigerator until required. If the sample has been refrigerated it must be stored at room temperature for at least four hours before the test is started. E+W

Day one E+W

11.  On day one tests must be carried out in duplicate using two 25 gram portions of each sample submitted for testing. Each 25 gram sample must be placed aseptically in a sterile container containing 225 ml Buffered Peptone Water/Lysine/Glucose (BPW/L/G) M15 and incubated at 37°C for 18 hours.

Marginal Citations

M15 Buffered Peptone Water/Lysine/Glucose – See Ogden I D (1988) International Journal of Food Microbiology 7:287-297, Elsevier Science BV, PO Box 211, 1000 AE, Amsterdam, Netherlands (ISSN 0168-1695).

Day two E+W

12.  On day two the incubated BPW/L/G must be added to Selenite Cystine Trimethylamine-N-Oxide Dulcitol (SC/T/D) M16 and Lysine Decarboxylase Glucose (LD/G) M17 media in electrical conductance cells or wells. For cells or wells containing more than 5 ml medium 0.2 ml of the BPW/L/G must be added and for cells or wells containing 5 ml or less medium 0.1 ml of the BPW/L/G must be added. Cells or wells must be connected to appropriate electrical conductance measuring equipment set to monitor and record changes in electrical conductance at 6 minute intervals over a 24 hour period. The temperature of cells and wells must be kept at 37°C.

Marginal Citations

M16 Selenite Cystine Trimethylamine-N-Oxide Dulcitol – See Easter, M C and Gibson, D M, (1985) Journal of Hygiene 94:245-262, Cambridge University Press, Cambridge.

M17 Lysine Decarboxylase Glucose- See Ogden I D (1988) International Journal of Food Microbiology 7:287-297, Elsevier Science BV, PO Box 211, 1000 AE, Amsterdam, Netherlands (ISSN 0168-1695).

Day three E+W

13.  On day three, at the end of the 24 hour period, the information recorded by the conductance measuring equipment must be analysed and interpreted using criteria defined by the manufacturers of the equipment. Where a well or cell is provisionally identified as being positive for Salmonella, the result must be confirmed by subculturing the contents of the well or cell on to two 90 millimetre plates of BGA or on to one 90 millimetre plate of BGA and one 90 millimetre plate of Xylose Lysine Deoxycholate Agar (XLD) using a 2.5 mm diameter loop. The plates must be inoculated with a droplet taken from the edge of the surface of the fluid by drawing the loop over the whole of one plate in a zig zag pattern and continuing to the second plate without recharging the loop. The space between the loop streaks must be 0.5 cm – 1.0 cm. The plates must be incubated at 37°C overnight.

Day four E+W

14.  On day four the plates must be examined and a minimum of 3 colonies from each plate showing suspicion of Salmonella growth must be subcultured–

(a) on to a blood agar plate;

(b) on to a MacConkey agar plate; and

(c) into biochemical media suitable for the identification of Salmonella.

These media must be incubated at 37°C overnight.

Day five E+W

15.  On day five the incubated composite media or equivalent must be examined and the findings recorded, discarding cultures which are obviously not Salmonella. Slide serological tests must be performed using Salmonella polyvalent “O”and polyvalent “H” (phase 1 and 2) agglutinating sera on selected suspect colonies collected from the blood agar or MacConkey plates. If reactions occur with one or both sera, the colonies must be typed by slide serology. If requested in writing by the National Assembly , the operator of the laboratory must send a subculture to a Regional Veterinary Laboratory of the Veterinary Laboratories Agency of the Department for Environment, Food and Rural Affairs for further typing.

PART III E+W METHOD FOR THE ISOLATION OF ENTEROBACTERIACEAE

1.  Tests must be begun on receipt of the sample or on the first working day which allows this method to be completed. If the test is not begun on the day of receipt the sample must be stored in a refrigerator until required at between 2°C and 8°C. If the sample has been refrigerated it must be removed from the refrigerator and stored at room temperature for at least one hour before the test is started. E+W

Samples E+W

2.  Tests must be carried out using five 10 gram portions of each sample submitted for testing. Each 10 gram sample must be placed aseptically in a sterile container containing 90 ml Buffered Peptone Water and mixed thoroughly until the sample is evenly suspended.

Inoculations E+W

3.  For each portion of the sample 1 ml of solution must be transferred to a sterile 90 mm petri dish (in duplicate). The plates must be labelled to identify the portion of sample they were taken from. 15 ml of Violet Red Bile Glucose Agar (VRBGA) M18 at a temperature of 47°C±2°C must be added to each petri dish and immediately gently mixed by swirling the dish with five clockwise and five anticlockwise circular movements.

Marginal Citations

M18 Violet Red Bile Glucose Agar – See Mossell D A A, Eelderink I, Koopmans M, van Rossem F (1978) Laboratory Practice 27 No. 12 1049-1050; Emap Maclaren, PO Box 109, Maclaren House, 19 Scarbrook Road, Croydon CR9 1QH.

4.  Once the agar has set, each agar plate must be overlaid with a further 10 ml VRBGA at a temperature of 47°C±2°C. Once the overlay has set, the plates must be inverted and incubated aerobically at 37°C±1°C for 20 hours±2 hours. E+W

Samples with colonies of Enterobacteriaceae E+W

5.  After incubation each set of duplicate plates must be examined for colonies characteristic of Enterobacteriaceae (purple colonies 1 – 2 mm in diameter). All characteristic colonies on each plate must be counted and the arithmetic mean of the duplicate plates taken.

The sample provisionally fails if either–

(a) any arithmetic mean is above 30 M19 ; or

(b) three or more arithmetic means are above 10;

in which case the following procedure must be followed to establish whether or not the colonies are Enterobacteriaceae.

Marginal Citations

M19 An arithmetic mean of 30 is equivalent to 3x102 colony forming units per gram of original sample.

6.  After counting the colonies, characteristic colonies must be taken at random from the agar plates, the number being at least the square root of the colonies counted. The colonies must be subcultured onto a blood agar plate and incubated aerobically at 37°C±1°C for 20 hours±2 hours. E+W

Examination of subcultures E+W

7.  An oxidase test and a glucose fermentation test must be performed on each of the five subcultured colonies. Colonies which are oxidase-negative and glucose fermentation-positive must be considered to be Enterobacteriaceae.

8.  If not all of the colonies prove to be Enterobacteriaceae, the total count in paragraph 5 must be reduced in proportion prior to establishing whether or not the sample should fail. E+W

Controls E+W

9.  Control tests must be carried out each day that a test is initiated using–

(a) Escherichia coli NCTC 10418 no more than seven days old at time of use; and

(b) processed animal protein or compost or digestion residue which is free of Enterobacteriaceae.

10.  A 10 gram portion of the rendered animal protein must be placed aseptically in a sterile container containing 90 ml BPW and mixed thoroughly until the sample is evenly suspended. E+W

11.  One colony of Escherichia coli must be placed in 10 ml BPW and mixed to form an even suspension. 0.1 ml of the suspension must be added to the suspension in the preceding paragraph. E+W

12.  This is then treated and examined in the same way as test samples. If no typical colonies are formed then that day's testing must be invalid and must be repeated. E+W